| Lignocellulose is one of the the most abundant renewable bioresource and low-cost biomass on the earth.Lignocellulosic biomass could be used as prime raw material for lipid producing due to its low cost.Xylose is the second-most concentrated sugar behind glucose in lignocellulosic hydrolyzate.Unefficient utilization of xylose is one of the biggest obstacles to commercial conversion of lignocellulose to lipid.Mucor circinelloides(M.circinelloides)is a lipid-accumulating model organism.It has been used as a production strain for γ-linolenic acid.Genomic sequence of M.circinelloides showed that it has xylose isomerase(XI)pathway but never been characterized.In this study,the xylose metabolism pathway and their effects on lipid synthesis in M.circinelloides were investigated to improve the industrial potential of M.circinelloides.The research work has been carried out as follow.The lipid production by M.circinelloides CBS 277.49,CBS 108.16 and WJ11 with xylose as carbon source were investigated respectively,and glucose was used in the control experiments.M.circinelloides exhibited similar biomass and lipid yield on xylose compare with those on glucose.Comparative analysis of the fermentation performance by M.circinelloides on different substrates revealed that xylose may be utilized in M.circinelloides through XI pathway.The xylA(encoding XI)and XKS1(encoding xylulosekinase(XK))genes were cloned from genomic cDNA of M.circinelloides 277.49.Then,the fragments were ligated to the vector pMAT1552 through the method of homologous recombination to construct expression vectors pMAT1552-xylA and p MAT1552-XKS1.The expression vectors were transformed into the pleu-MU402(uridine auxotrophic strain)to get the recombinant strains Mc-XI and Mc-XK.The plasmid pMAT1552 was also transformed into pleu-MU402 to get the recombinant Mc-1552 as the control strain.Mc-XI,Mc-XK and Mc-1552 were grown in lipid accumulation medium with 50 g/L xylose.The result showed that overexpressing XI or XK increased the total consumption rate of xylose and lipid yield.In the lipid accumulation phase,the mRNA levels of XI and XK genes,and activities of XI,XK,glucose 6-phosphatedehydrogenase(G6PDH)and 6-phosphogluconate dehydrogenase(6PGDH)in recombinant stains were higher than that of the control.Mc-XI and Mc-XK were cultivated in lipid accumulation medium containing glucose(33 g/L)and xylose(17 g/L)as the carbon source.The results showed that overexpression of XI and XK contributed to alleviate the glucose repression.Compared with Mc-1552,the consumption rate of xylose in Mc-XI and Mc-XK increased by 37% and 25%,respectively.The lipid content in Mc-XI and Mc-XK increased by 8% and 28% compared with the control.In addition,the analysis of transcriptional level and enzyme activity demonstrated that the presence of glucose inhibited the expression of XK to a certain extent.The xylA and XKS1 were fusion-expressed in M.circinelloides by designing a fragment consisting of xylA and XKS1.The fragment was ligated to pMAT1552 to construct the expression vectors pMAT1552-xylA+XKS1.Subsequently,pMAT1552-xylA+XKS1 was transformed into the pleu-MU402 to get the recombinant strains Mc-OL.Compared with Mc-1552,Mc-OL could significantly increase the consumption rate of xylose on the substrate of xylose or mixted sugars,while the lipid yield was not increased.The reason for this result may be due to that the excessive consumption of ATP and the high level accumulation of xylulose-5-phosphate in Mc-OL,which was harmful to the growth of M.circinelloides and eventually blocked the synthesis of lipid. |
PDF Full Text Request